Calcium plays many roles in the signal-transduction cascade in olfactory receptor neurons (ORNs). Ca2+ enters through cAMP-gated channels in response to odor-ligand receptor activation of G-protein-mediated increases in adenylyl cyclase activity. Ca2+ entry is a key step in mammalian olfactory chemosensory transduction and stimulates a Ca2+-activated chloride channel to amplify the transduction current. Ca2+ is also a regulator of adaptation, desensitization, recovery and transduction-translation coupling. Much effort has been directed towards understanding these events in the ORN. Much less attention has been paid to downstream events that maintain stable intracellular Ca2+ levels in ORNs. Our characterization of the olfactory marker protein (OMP)-null mouse phenotype using electrophysiological, biochemical and behavioral measures in the previous funding cycle have led us to hypothesize that the OMP-null phenotvpe is due to a defect in regulating intracellular Ca2+ levels. Specifically. we hypothesize that OMP participates in regulating the activity of Ca extrusion processes in ORNs. The specificity of OMP expression in mature ORNs, its sequence conservation across all vertebrates, and the OMP-null mouse phenotype all indicate OMP is important to olfactory function. We recently observed a significant alteration in Ca2+ kinetics in ORN dendritic knobs in OMP-null mice following stimulation. Several genes participate in Ca2+ extrusion including the Na+/Ca2+ exchangers (NCX), and the Na+/Ca2+-K+ exchangers (NCKX). Each of these is represented by three genes and numerous mRNA splice variants expressed in cell- and tissue-specific patterns. Immunocytochemical and electrophysiological reports suggest that these exchangers are important in maintaining intracellular ORN Ca2+ levels. These reports, coupled with our new data that OMP modulates Ca2+ extrusion further supports our hypothesis and the importance of characterizing the molecular and biochemical bases of Ca2+ extrusion processes in ORNs. This is a significant gap in our knowledge that this proposal addresses. We will (l)-characterize NCX and NCKX gene expression in olfactory tissue, and especially in ORNs, during development and in response to lesions; (2)-identify that part of the specific NCX or NCKX protein that participates in the OMP-mediated regulation of Ca + levels; (3)-determine the nature and function of other participants in this process e.g. the recently discovered Bex proteins that bind to OMP. These studies will provide novel insights to the mechanisms by which intracellular Ca2+ levels are regulated in ORNs and may generate chemotherapeutic opportunities for treatment of anosmias or dysosmias. bERFORMANCE SITE(S) (organization, city, State) KEY PERSONNEL, see instructions on Page 11. Use continuation pages as needed to provide the required information in the format shown below. Name Organization Role on Project Frank L Margolis University of Maryland PI Joyce W. Margolis University of Maryland Research Specialist Jae Hyung Koo University of Maryland Post Doc Martina M. Pyrski University of Maryland Post Doc Kyrill Ukhanov University of Maryland Post Doc Dan Schulze University of Maryland Collaborator Donna M. Baldisseri University of Maryland Collaborator Abdul M. Ruknudin University of Maryland Collaborator Trese Leinders-Zufall University of Maryland Consultant PHS 398 (Rev. 4/98) Page 2 BB Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. rnncipai n^ptigaior/rrogram uirecior (Last, nrsi, miaa/ey. ^md^Miib, rimus. Type the name of the principal investigator/programdirector at the top of each printed page and each continuation page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page [unreadable]. 1 Description,